Use of akkermansia for treating metabolic disorders

ABSTRACT

The present invention relates to Akkermansia muciniphila or fragments thereof for treating a metabolic disorder in a subject in need thereof. The present invention also relates to a composition, a pharmaceutical composition and a medicament comprising Akkermansia muciniphila or fragments thereof for treating a metabolic disorder. The present invention also relates to the use of Akkermansia muciniphila or fragments thereof for promoting weight loss in a subject in need thereof.

RELATED APPLICATIONS

This application is a continuation of U.S. Nonprovisional patentapplication Ser. No. 14/443,829, filed May 19, 2015, which is a 35U.S.C. § 371 U.S. national stage entry of International PatentApplication No. PCT/EP2013/073972, filed Nov. 15, 2013, which claimspriority to PCT/EP2012/073011, filed Nov. 19, 2012.

FIELD OF INVENTION

The present invention relates to the treatment of metabolic disorders,such as, for example, metabolic disorders related to overweight andobesity, such as, for example, Diabetes Mellitus or high cholesterol.The present invention more specifically relates to a compositioncomprising Akkermansia spp or fragments thereof for treating a metabolicdisorder.

BACKGROUND OF INVENTION

Obesity is a worldwide problem, with an estimated number of obese adultsof about 250 million. This epidemic of obesity is correlated with agreat increase in the prevalence of obesity-related disorders, such as,for example, Diabetes, hypertension, cardiac pathologies and liverdiseases. Due to these highly disabling pathologies, obesity iscurrently considered in western countries as one of the most importantpublic health problem. There is thus a real need of compositions andmethods for treating or preventing obesity and/or obesity-relateddisorders.

Obesity and obesity-related diseases are associated with (i) metabolicdysfunctions (with an impact on glucose homeostasis and lipid metabolismfor example); (ii) low grade inflammatory state associated to higherblood lipopolysaccharides (LPS) levels (also referred as metabolicendotoxemia); and (iii) impaired gut barrier function (i.e. increasedgut permeability). In order to treat obesity, impact on at least one,preferably 2 and more preferably 3 of these 3 factors is thus needed.

The human gut is colonized by a diverse, complex and dynamic communityof microbes representing over 1000 different species, which continuouslyinteract with the host (Zoetendal, Rajilic-Stojanovic and de Vos, Gut2008, 57: 1605-1615). The homeostasis of the gut microbiota is dependenton host characteristics (age, gender, genetic background . . . ) andenvironmental conditions (stress, drugs, gastrointestinal surgery,infectious and toxic agents . . . ), but also on the day-to-day dietarychanges. Growing evidences support the role of gut microbiota in thedevelopment of obesity and related disorders (Delzenne & Cani, Annu.Rev. Nutr. 2011, 31: 15-31).

Therefore, treatment with products that target the gut microbiotaappeared as promising therapeutic tools for treating obesity and relateddisorders. These products may consist of living microbes, such as in thecase of most probiotics, or contain dead microbes or fragments thereof.In addition, these products may comprise substrates that are used by thegut microbiota, such as in the case of prebiotics, or contain compoundsthat change the balance of the intestinal microbiota, such as specificantimicrobial compounds.

For example, WO 2008/076696 describes the gut microbiota as atherapeutic target for treating obesity and related disorders. WO2008/076696 specifically describes methods for altering the abundance ofBacteroides and/or Firmicutes in the gut of a subject, by administeringantibiotics and/or probiotics to the subject.

Moreover, EP 2 030 623 relates to the prevention and/or treatment ofmetabolic disorders, such as, for example, obesity related disorders, byregulating the amount of Enterobacteria in the gut. EP 2 030 623discloses reducing the amount of Enterobacteria in the gut byadministering probiotic bacteria, such as, for example, Bifidobacterium,Lactococcus, Streptococcus, Enterococcus or Lactobacillus.

Furthermore, the Applicant described that the gut microbiota is modifiedin prebiotic-treated obese mice (Everard et al., Diabetes, 2011November; 60(11):2775-86). Moreover, prebiotics (1) improve glucose andlipid metabolisms in obese mice, (2) reduce plasma LPS and improve gutbarrier function (e.g. reduction of inflammation) in obese mice, (3)induce an increased enteroendocrine L-cell number in obese mice, and (4)improve leptin sensitivity and glucose homeostasy in diet-induced obeseand diabetic mice.

Among the modification induced by prebiotic treatment of obese mice is aconsiderable alteration of the gut microbiota composition, characterizedby (i) a decreased abundance of Bacteroidetes, Lactobacillus spp andbacteria of the Bacteroides-Prevotella group; and (ii) an increasedabundance of Bifidobacterium spp., of bacteria of the E. rectale/C.coccoides group and of Akkermansia muciniphila, belonging to theVerrucomicrobia. A. muciniphila, a bacteria firstly identified in 2004by the Applicant, represents approximately 1 to 3% of the totalmicrobiota of healthy adults (Derrien et al, International Journal ofSystematic and Evolutionary Microbiology, 2004, 54:1469-1476; Derrien etal Applied Environmental Microbiology 2008, 74, 1646-1648.).

Indirect evidences suggested a relationship between the abundance of A.muciniphila and intestinal dysfunctions or obesity-related disorders.For example, WO 2011/107481 describes that the absence of Akkermansiamuciniphila in the gut of a subject, combined with the presence ofBacteroides capillosus and Clostridium leptum indicates that thissubject is suffering from ulcerative colitis. Moreover, Hansen andcolleagues showed that the administration of an antibiotic, Vancomycin,to neonatal NOD mice (the NOD mouse model is a model for Diabetes)suppresses clinical onset of Diabetes and propagates Akkermansiamuciniphila (Hansen et al., Diabetologia, 2012 August; 55(8):2285-94).However, this may be an indirect effect due to the insensitivity ofintestinal Akkermansia spp. to the used antibiotic.

These results thus showed that the complete composition of the gutmicrobiota is modified following the administration of prebiotic inmice. More specifically, no evidence suggested a specific role of onebacterial species, such as, for example, Akkermansia muciniphila, in thebeneficial response to prebiotics administration. Moreover, to theApplicant knowledge, no beneficial effect of the direct administrationof Akkermansia muciniphila has never been described, nor suggested.

Here, the Applicant surprisingly showed that repeated administration ofAkkermansia muciniphila alone impacts the three underlying dysfunctionsassociated with obesity and related disorders, i.e. metabolicdysfunctions, low grade inflammatory state associated to higher bloodlipopolysaccharides (LPS) levels and impaired gut barrier function. Thepresent invention thus relates to the use of Akkermansia muciniphila orfragments thereof for treating obesity and related disorders.

SUMMARY

The present invention thus relates to Akkermansia muciniphila orfragments thereof for treating, or for use in treating, a metabolicdisorder in a subject in need thereof. In one embodiment of theinvention, said metabolic disorder is obesity. In another embodiment ofthe invention, said metabolic disorder is selected from the groupcomprising metabolic syndrome, insulin-deficiency or insulin-resistancerelated disorders, Diabetes Mellitus (such as, for example, Type 2Diabetes), glucose intolerance, abnormal lipid metabolism,atherosclerosis, hypertension, cardiac pathology, stroke, non-alcoholicfatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia,dysfunction of the immune system associated with overweight and obesity,cardiovascular diseases, high cholesterol, elevated triglycerides,asthma, sleep apnoea, osteoarthritis, neuro-degeneration, gallbladderdisease, syndrome X, inflammatory and immune disorders, atherogenicdyslipidemia and cancer.

The present invention also relates to Akkermansia muciniphila orfragments thereof for increasing energy expenditure of a subject,preferably without impacting the food intake of said subject. Thepresent invention also relates to Akkermansia muciniphila or fragmentsthereof for increasing satiety in a subject.

In one embodiment of the invention, viable cells of Akkermansiamuciniphila are administered to the subject in need thereof.

In one embodiment of the invention, Akkermansia muciniphila is orallyadministered.

In one embodiment of the invention, an amount of Akkermansia muciniphilaranging from about 1.10² to about 1.10¹⁵ cfu, preferably from about1.10⁴ to about 1.10¹² cfu, more preferably from about 1.10⁵ to about1.10¹⁰ cfu, and even more preferably from about 1.10⁶ to about 1.10⁹ cfuis administered to the subject. In another embodiment of the invention,an amount of Akkermansia muciniphila ranging from about 1.10⁴ to about1.10¹² cfu, more preferably from about 1.10⁵ to about 1.10¹¹ cfu, andeven more preferably from about 1.10⁶ to about 1.10¹⁰ cfu isadministered to the subject.

In one embodiment of the invention, Akkermansia muciniphila isadministered at least once a day. In one embodiment of the invention,Akkermansia muciniphila is administered at least three times a week. Inanother embodiment of the invention, Akkermansia muciniphila isadministered at least once a week.

In one embodiment of the invention, Akkermansia muciniphila isco-administered with another probiotic strain and/or with one or moreprebiotics.

Another object of the invention is a composition for treating, or foruse in treating, a metabolic disorder, or for increasing energyexpenditure or for increasing satiety in a subject comprisingAkkermansia muciniphila or fragments thereof as described hereinabove inassociation with an excipient. In one embodiment, said composition is anutritional composition. In one embodiment of the invention, saidcomposition is orally administered.

The present invention also relates to a pharmaceutical composition fortreating, or for use in treating, a metabolic disorder or for increasingenergy expenditure or for increasing satiety in a subject comprisingAkkermansia muciniphila or fragments thereof as hereinabove described inassociation with a pharmaceutically acceptable vehicle.

Another object of the present invention is a medicament for treating, orfor use in treating, a metabolic disorder or for increasing energyexpenditure or for increasing satiety in a subject comprisingAkkermansia muciniphila or fragments thereof as hereinabove described.

Another object of the present invention is the use of Akkermansiamuciniphila or fragments thereof for promoting weight loss in a subjectin need thereof.

The present invention also relates to a cosmetic composition comprisingAkkermansia muciniphila or fragments thereof for promoting weight lossin a subject in need thereof.

Definitions

In the present invention, the following terms have the followingmeanings:

-   -   “Treatment” means preventing (i.e. keeping from happening),        reducing or alleviating at least one adverse effect or symptom        of a disease, disorder or condition. This term thus refers to        both therapeutic treatment and prophylactic or preventative        measures; wherein the object is to prevent or slow down (lessen)        the targeted pathologic condition or disorder. In one embodiment        of the invention, those in need of treatment include those        already with the disorder as well as those prone to have the        disorder or those in whom the disorder is to be prevented.    -   “Effective amount” refers to level or amount of agent that is        aimed at, without causing significant negative or adverse side        effects to the target, (1) delaying or preventing the onset of a        metabolic disorder; (2) slowing down or stopping the        progression, aggravation, or deterioration of one or more        symptoms of the metabolic disorder; (3) bringing about        ameliorations of the symptoms of the metabolic disorder; (4)        reducing the severity or incidence of the metabolic        disorder; (5) curing the metabolic disorder; or (6) restoring        the normal amount and/or proportion of Akkermansia muciniphila        in the gut of the subject to be treated. An effective amount may        be administered prior to the onset of a metabolic disorder, for        a prophylactic or preventive action. Alternatively or        additionally, the effective amount may be administered after        initiation of the metabolic disorder, for a therapeutic action.    -   “Akkermansia muciniphila” refers to the strictly anaerobic        mucin-degrading bacteria identified by Derrien (Derrien et al,        International Journal of Systematic and Evolutionary        Microbiology, 2004, 54:1469-1476). Cells are oval-shaped,        non-motile and stain Gram-negative. Akkermansia muciniphila may        also be referred as Akkermansia spp. or Akkermansia-like        bacteria. It belongs to the Chlamydiae/Verrucomicrobia group;        Verrucomicrobia phylum. If the taxonomy should change, the        skilled artisan would know how to adapt the changes in the        taxonomy to deduce the strains that could be used in the present        invention. Moreover, the complete genome of Akkermansia        muciniphila has been determined by the Applicant (van Passel et        al, PLoS One 6, 2011: e16876). It is generally accepted that        strains with a genome similarity of about 70% can be considered        as the same species.    -   “Probiotics” refers to microbial cell preparations (such as, for        example, living microbial cells) or components of microbial        cells which, when administered in an effective amount, provide a        beneficial effect on the health or well-being of a subject. By        definition, all probiotics have a proven non-pathogenic        character. In one embodiment, these health benefits are        associated with improving the balance of human or animal        microbiota in the gastro-intestinal tract, and/or restoring        normal microbiota.    -   “Prebiotic” refers to a substance, such as, for example, a food        substance, which may not be digested by humans, but which may be        used by bacteria of the gut microbiota and which is intended to        promote the growth of probiotic bacteria in the intestine.    -   “Overweight” refers to a subject situation wherein said subject        has a Body Mass Index (BMI) ranging from 25 to 30. As used        herein, BMI is defined as the individual's body mass (in kg)        divided by the square of his/her height (in meter). “Obesity”        refers to a subject situation wherein said subject has a BMI        superior or equal to 30.    -   “Subject” refers to an animal, preferably a mammal, more        preferably a human. In one embodiment, the subject is a male. In        another embodiment, the subject is a female. In one embodiment        of the invention, a subject may also refer to a pet, such as,        for example, a dog, a cat, a guinea pig, a hamster, a rat, a        mouse, a ferret, a rabbit and the like.    -   “About” preceding a figure means plus or less 20%, preferably        10% of the value of said figure.    -   “Fragment” may refer to cellular components, metabolites,        secreted molecules and compounds resulting from the metabolism        of Akkermansia muciniphila and the like. Fragments may be        obtained, for example, by recovering the supernatant of a        culture of Akkermansia muciniphila or by extracting cell        components or cell fractions, metabolites or secreted compounds        from a culture of Akkermansia muciniphila. The term fragment may        also refer to a degradation product. A fragment may correspond        to a component in the isolated form or to any mixture of one or        more components derived from Akkermansia muciniphila. In one        embodiment, a fragment may correspond to one or more of such a        components present in Akkermansia muciniphila that is produced        in another way, such as using recombinant DNA technology, in a        microbial host or in any other (bio)synthetic process.    -   “Metabolic disorder” refers to disorders, diseases and        conditions caused or characterized by abnormal weight gain,        energy use or consumption, altered responses to ingested or        endogenous nutrients, energy sources, hormones or other        signaling molecules within the body or altered metabolism of        carbohydrates, lipids, proteins, nucleic acids or a combination        thereof. A metabolic disorder may be associated with either a        deficiency or an excess in a metabolic pathway resulting in an        imbalance in metabolism of carbohydrates, lipids, proteins        and/or nucleic acids. Examples of metabolic disorders include,        but are not limited to, metabolic syndrome, insulin-deficiency        or insulin-resistance related disorders, Diabetes Mellitus (such        as, for example, Type 2 Diabetes), glucose intolerance, abnormal        lipid metabolism, atherosclerosis, hypertension, cardiac        pathology, stroke, non-alcoholic fatty liver disease,        hyperglycemia, hepatic steatosis, dyslipidemia, dysfunction of        the immune system associated with overweight and obesity,        cardiovascular diseases, high cholesterol, elevated        triglycerides, asthma, sleep apnoea, osteoarthritis,        neuro-degeneration, gallbladder disease, syndrome X,        inflammatory and immune disorders, atherogenic dyslipidemia and        cancer.

DETAILED DESCRIPTION

This invention relates to Akkermansia muciniphila or a fragment thereoffor treating, or for use in treating, metabolic disorders in a subjectin need thereof.

As used herein, a metabolic disorder is a disorder related to an alteredmetabolic homeostasis, such as, for example, an altered glucidic orlipidic homeostasis.

In one embodiment of the invention, said metabolic disorder is obesity.

Examples of other metabolic disorders include, but are not limited to,metabolic syndrome, insulin-deficiency or insulin-resistance relateddisorders, Diabetes Mellitus (such as, for example, Type 2 Diabetes),glucose intolerance, abnormal lipid metabolism, atherosclerosis,hypertension, cardiac pathology, stroke, non-alcoholic fatty liverdisease, hyperglycemia, hepatic steatosis, dyslipidemia, dysfunction ofthe immune system associated with overweight and obesity, cardiovasculardiseases, high cholesterol, elevated triglycerides, asthma, sleepapnoea, osteoarthritis, neuro-degeneration, gallbladder disease,syndrome X, inflammatory and immune disorders, atherogenic dyslipidemiaand cancer.

In another embodiment, said metabolic disorder is an overweight and/orobesity related metabolic disorder, i.e. a metabolic disorder that maybe associated to or caused by overweight and/or obesity. Examples ofoverweight and/or obesity related metabolic disorder include, but arenot limited to metabolic syndrome, insulin-deficiency orinsulin-resistance related disorders, Diabetes Mellitus (such as, forexample, Type 2 Diabetes), glucose intolerance, abnormal lipidmetabolism, atherosclerosis, hypertension, cardiac pathology, stroke,non-alcoholic fatty liver disease, hyperglycemia, hepatic steatosis,dyslipidemia, dysfunction of the immune system associated withoverweight and obesity, cardiovascular diseases, high cholesterol,elevated triglycerides, asthma, sleep apnoea, osteoarthritis,neuro-degeneration, gallbladder disease, syndrome X, inflammatory andimmune disorders, atherogenic dyslipidemia and cancer.

In one embodiment, said metabolic disorder is Diabetes Mellitus,preferably Type 2 Diabetes. In another embodiment, said metabolicdisorder is hypercholesterolemia (also known as high cholesterol). Inone embodiment, hypercholesterolemia corresponds to a plasma cholesterolconcentration superior or equal to 2 g/L or 5 mmol/L. In anotherembodiment, hypercholesterolemia corresponds to a ratio plasmaconcentration of total cholesterol:plasma concentration of HDL (highdensity lipoprotein cholesterol) superior or equal to 4.5:1, preferably5:1.

In one embodiment of the invention, living strains of Akkermansiamuciniphila are used in the present invention, preferably the livingstrains are derived from cells in stationary phase of growth.

In one embodiment of the invention, Akkermansia muciniphila may be inthe form of viable cells. In another embodiment of the invention,Akkermansia muciniphila may be in the form of non-viable cells.

In one embodiment, metabolically active Akkermansia muciniphila cellsare used in the present invention. In one embodiment, strains ofAkkermansia muciniphila are not metabolically inactivated, whereinmetabolic inactivation may result, for example, from autoclavetreatment.

In one embodiment, Akkermansia muciniphila or fragment thereof issubstantially purified. As used herein, the term “substantiallypurified” means that Akkermansia muciniphila or fragment thereof iscomprised in a sample wherein it represents at least about 50%,preferably at least about 60, 70, 80, 85, 90, 95, 99% or more of thebacterial strains or fragment thereof of said sample.

The present invention also relate to a composition comprising aneffective amount of Akkermansia muciniphila or a fragment thereof fortreating, or for use in treating, a metabolic disorder.

In one embodiment of the invention, the effective amount of Akkermansiamuciniphila corresponds to the amount of the bacteria sufficient forrestoring a normal amount and/or proportion of Akkermansia muciniphilawithin the gut of the subject. Indeed, the Applicant showed that the gutof obese or overweight subject is depleted in Akkermansia muciniphila(see Examples). In one embodiment of the invention, the normal amountand/or proportion of Akkermansia muciniphila corresponds to the amount,and/or to the proportion of Akkermansia muciniphila present in the gutof a healthy subject.

As used herein, the term “healthy subject” is used to define a subjectwhich is not affected by the disease to be treated. For example, ifAkkermansia muciniphila or a fragment thereof is used for treatingobesity, the healthy subject is not affected by obesity. Preferably, thehealthy subject shares common characteristics with the subject to betreated, such as, for example, same gender, age, sex, diet, drugs intakeor geolocation.

In one embodiment of the invention, the normal proportion of Akkermansiamuciniphila in the gut ranges from about 0.1% to about 10% (in number ofAkkermansia muciniphila cells to the total number of bacteria cells ofthe gut), preferably from about 0.3% to about 5%, more preferably fromabout 1% to about 3%.

In one embodiment of the invention, the effective amount of Akkermansiamuciniphila ranges from about 1.10² to about 1.10¹⁵ cfu, preferably fromabout 1.10⁴ to about 1.10¹² cfu, more preferably from about 1.10⁵ toabout 1.10¹⁰ cfu, and even more preferably from about 1.10⁶ to about1.10⁹ cfu, wherein cfu stands for “colony forming unit”.

In another embodiment of the invention, the effective amount ofAkkermansia muciniphila ranges from about 1.10⁶ to about 1.10¹⁰ cfu,preferably from about 1.10⁸ to about 1.10¹⁰ cfu, more preferably fromabout 1.10⁹ to about 1.10¹⁰ cfu.

In another embodiment of the invention, the effective amount ofAkkermansia muciniphila ranges from about 1.10⁶ to about 1.10¹⁰ cfu,preferably from about 1.10⁶ to about 1.10⁹ cfu, more preferably fromabout 1.10⁸ to about 1.10⁹ cfu.

In one embodiment of the invention, the effective amount of a fragmentof Akkermansia muciniphila ranges from fragments derived from about1.10² to about 1.10¹⁵ cfu, preferably from about 1.10⁴ to about 1.10¹²cfu, more preferably from about 1.10⁵ to about 1.10¹⁰ cfu, and even morepreferably from about 1.10⁶ to about 1.10⁹ cfu, wherein cfu stands for“colony forming unit”. In another embodiment of the invention, theeffective amount of a fragment of Akkermansia muciniphila ranges fromfragments derived from about 1.10⁶ to about 1.10¹⁰ cfu, preferably fromabout 1.10⁸ to about 1.10¹⁰ cfu, more preferably from about 1.10⁹ toabout 1.10¹⁰ cfu. In another embodiment of the invention, the effectiveamount of a fragment of Akkermansia muciniphila ranges from fragmentsderived from about 1.10⁶ to about 1.10¹⁰ cfu, preferably from about1.10⁶ to about 1.10⁹ cfu, more preferably from about 1.10⁸ to about1.10⁹ cfu.

In one embodiment of the invention, the composition of the inventioncomprises an amount of Akkermansia muciniphila ranging from about 1.10²to about 1.10¹⁵ cfu/g of the composition, preferably from about 1.10⁴ toabout 1.10¹² cfu/g of the composition, more preferably from about 1.10⁵to about 1.10¹⁰ cfu/g of the composition and even more preferably fromabout 1.10⁶ to about 1.10⁹ cfu/g of the composition. In one embodimentof the invention, the composition of the invention comprises an amountof Akkermansia muciniphila ranging from about 1.10² to about 1.10¹⁵cfu/mL of the composition, preferably from about 1.10⁴ to about 1.10¹²cfu/mL of the composition, more preferably from about 1.10⁵ to about1.10¹⁰ cfu/mL of the composition and even more preferably from about1.10⁶ to about 1.10⁹ cfu/mL of the composition. In another embodiment ofthe invention, the composition of the invention comprises an amount ofAkkermansia muciniphila ranging from about 1.10⁶ to about 1.10¹⁰ cfu/gor cfu/mL of the composition, preferably from about 1.10⁸ to about1.10¹⁰ cfu/g or cfu/mL, more preferably from about 1.10⁹ to about 1.10¹⁰cfu/g or cfu/mL. In another embodiment of the invention, the compositionof the invention comprises an amount of Akkermansia muciniphila rangingfrom about 1.10⁶ to about 1.10¹⁰ cfu/g or cfu/mL of the composition,preferably from about 1.10⁶ to about 1.10⁹ cfu/g or cfu/mL, morepreferably from about 1.10⁸ to about 1.10⁹ cfu/g or cfu/mL.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragments of Akkermansia muciniphila ranging fromfragments derived from about 1.10² to about 1.10¹⁵ cfu/g or mL of thecomposition, preferably from about 1.10⁴ to about 1.10¹² cfu/g or mL ofthe composition, more preferably from about 1.10⁵ to about 1.10¹⁰ cfu/gor mL of the composition and even more preferably from about 1.10⁶ toabout 1.10⁹ cfu/g or mL of the composition. In another embodiment of theinvention, the composition of the invention comprises an amount offragments of Akkermansia muciniphila ranging from fragments derived fromabout 1.10⁶ to about 1.10¹⁰ cfu/g or cfu/mL of the composition,preferably from about 1.10⁸ to about 1.10¹⁰ cfu/g or cfu/mL, morepreferably from about 1.10⁹ to about 1.10¹⁰ cfu/g or cfu/mL. In anotherembodiment of the invention, the composition of the invention comprisesan amount of fragments of Akkermansia muciniphila ranging from fragmentsderived from about 1.10⁶ to about 1.10¹⁰ cfu/g or cfu/mL of thecomposition, preferably from about 1.10⁶ to about 1.10⁹ cfu/g or cfu/mL,more preferably from about 1.10⁸ to about 1.10⁹ cfu/g or cfu/mL.

The present invention also relates to a pharmaceutical compositioncomprising an effective amount of Akkermansia muciniphila or a fragmentthereof and at least one pharmaceutically acceptable excipient. In oneembodiment of the invention, the pharmaceutical composition of theinvention is for treating a metabolic disorder. In another embodiment ofthe invention, the pharmaceutical composition is for restoring a normalproportion of Akkermansia muciniphila in the gut of a subject in needthereof.

As used herein the term “pharmaceutically acceptable excipient” refersto an excipient that does not produce an adverse, allergic or otheruntoward reaction when administered to an animal, preferably a human. Itmay include any and all solvents, dispersion media, coatings, isotonicand absorption delaying agents and the like. For human administration,preparations should meet sterility, pyrogenicity, general safety andpurity standards as required by FDA Office of Biologics standards.

The present invention also relates to a medicament comprising aneffective amount of Akkermansia muciniphila or a fragment thereof. Inone embodiment of the invention, the medicament of the invention is fortreating a metabolic disorder. In another embodiment of the invention,the medicament is for restoring a normal proportion of Akkermansiamuciniphila in the gut of a subject in need thereof.

The present invention also relates to a method for treating a metabolicdisorder in a subject in need thereof, wherein said method comprisesadministering an effective amount of Akkermansia muciniphila or afragment thereof to the subject.

Another object of the invention is a method for restoring a normalproportion of Akkermansia muciniphila in the gut of a subject in needthereof, wherein said method comprises administering an effective amountof Akkermansia muciniphila or a fragment thereof to the subject.

In one embodiment, the method of the invention comprises administeringan effective amount of the composition, of the pharmaceuticalcomposition or of the medicament of the invention to the subject.

In one embodiment of the invention, Akkermansia muciniphila or afragment thereof, or the composition, pharmaceutical composition ormedicament is administered at least once a week, preferably at leasttwice a week, more preferably at least three times a week, and even morepreferably three times a week. In another embodiment, Akkermansiamuciniphila or a fragment thereof, or the composition, pharmaceuticalcomposition or medicament is administered at least once a day, andpreferably at least twice a day.

In one embodiment, Akkermansia muciniphila or a fragment thereof, or thecomposition, pharmaceutical composition or medicament of the inventionis administered during 1 week, preferably during 2, 3, 4, 5, 6, 7 or 8weeks or more.

In one embodiment, Akkermansia muciniphila or a fragment thereof, or thecomposition, pharmaceutical composition or medicament of the inventionis administered for a period that lasts until the desired outcome isachieved (e.g. weight loss, metabolic disorder treatment, decrease ofcholesterol plasma level . . . ).

In one embodiment, the administration of Akkermansia muciniphila or afragment thereof, or the composition, pharmaceutical composition ormedicament of the invention is permanent, i.e. is not limited in time.

In one embodiment of the invention, the daily amount of Akkermansiamuciniphila administered per day ranges from 1.10² to about 1.10¹⁵cfu/day, preferably from about 1.10⁴ to about 1.10¹² cfu/day, morepreferably from about 1.10⁵ to about 1.10¹⁰ cfu/day and even morepreferably from about 1.10⁶ to about 1.10⁹ cfu/day.

In another embodiment of the invention, the daily amount of Akkermansiamuciniphila administered per day ranges from 1.10⁶ to about 1.10¹⁰cfu/day, preferably from about 1.10⁸ to about 1.10¹⁰ cfu/day, morepreferably from about 1.10⁹ to about 1.10¹⁰ cfu/day.

In another embodiment of the invention, the daily amount of Akkermansiamuciniphila administered per day ranges from 1.10⁶ to about 1.10¹⁰cfu/day, preferably from about 1.10⁶ to about 1.10⁹ cfu/day, morepreferably from about 1.10⁸ to about 1.10⁹ cfu/day.

In one embodiment of the invention, the daily amount of fragments ofAkkermansia muciniphila administered per day ranges from fragmentsderived from 1.10² to about 1.10¹⁵ cfu/day, preferably from about 1.10⁴to about 1.10¹² cfu/day, more preferably from about 1.10⁵ to about1.10¹⁰ cfu/day and even more preferably from about 1.10⁶ to about 1.10⁹cfu/day. In another embodiment of the invention, the daily amount offragments of Akkermansia muciniphila administered per day ranges fromfragments derived from 1.10⁶ to about 1.10¹⁰ cfu/day, preferably fromabout 1.10⁸ to about 1.10¹⁰ cfu/day, more preferably from about 1.10⁹ toabout 1.10¹⁰ cfu/day. In another embodiment of the invention, the dailyamount of fragments of Akkermansia muciniphila administered per dayranges from fragments derived from 1.10⁶ to about 1.10¹⁰ cfu/day,preferably from about 1.10⁶ to about 1.10⁹ cfu/day, more preferably fromabout 1.10⁸ to about 1.10⁹ cfu/day.

In one embodiment of the invention, the subject is overweight. Inanother embodiment, the subject is obese.

In one embodiment of the invention, the subject is diagnosed with ametabolic disorder, such as, for example, with an overweight and/orobesity related metabolic disorder.

In another embodiment, the subject is at risk of developing a metabolicdisorder, such as, for example, an overweight and/or obesity relatedmetabolic disorder. In one embodiment, said risk is related to the factthat the subject is overweight or obese. In another embodiment, saidrisk corresponds to a predisposition, such as, for example, a familialpredisposition to a metabolic disorder, such as, for example, to anoverweight and/or obesity related metabolic disorder.

In one embodiment of the invention, the subject presents a deregulationof the gut microbiota composition. Preferably, the gut microbiota ofsaid subject is depleted in Akkermansia muciniphila strains. In oneembodiment, the proportion of Akkermansia muciniphila in the gut of thesubject is inferior to 1%, preferably inferior to 0.5%, more preferablyinferior to 0.1%, in number of Akkermansia muciniphila cells to thetotal number of bacterial cells in the gut.

The present invention also relates to the cosmetic use of Akkermansiamuciniphila or a fragment thereof for promoting weight loss in asubject.

Another object of the invention is thus a cosmetic compositioncomprising a cosmetically effective amount of Akkermansia muciniphila ora fragment thereof, and the use thereof for promoting weight loss in asubject. As used herein, a “cosmetically effective amount” refers to theamount of a cosmetic composition necessary and sufficient for promotinga cosmetic effect, such as, for example, for inducing weight loss in asubject.

The present invention also relates to a method for promoting weight lossin a subject in need thereof, wherein said method comprisesadministering a cosmetically effective amount of Akkermansia muciniphilaor a fragment thereof to said subject.

In one embodiment, the method of the invention comprises administering acosmetically effective amount of the composition or of the cosmeticcomposition of the invention to the subject.

In one embodiment of the invention, the cosmetically effective amount ofAkkermansia muciniphila ranges from about 1.10² to about 1.10¹⁵ cfu,preferably from about 1.10⁴ to about 1.10¹² cfu, more preferably fromabout 1.10⁵ to about 1.10¹⁰ cfu and even more preferably from about1.10⁶ to about 1.10⁹ cfu. In another embodiment of the invention, thecosmetically effective amount of Akkermansia muciniphila ranges fromabout 1.10⁶ to about 1.10¹⁰ cfu, preferably from about 1.10⁸ to about1.10¹⁰ cfu, more preferably from about 1.10⁹ to about 1.10¹⁰ cfu. Inanother embodiment of the invention, the cosmetically effective amountof Akkermansia muciniphila ranges from about 1.10⁶ to about 1.10¹⁰ cfu,preferably from about 1.10⁶ to about 1.10⁹ cfu, more preferably fromabout 1.10⁸ to about 1.10⁹ cfu.

In one embodiment of the invention, the cosmetically effective amount offragments of Akkermansia muciniphila ranges from fragments derived fromabout 1.10² to about 1.10¹⁵ cfu, preferably from about 1.10⁴ to about1.10¹² cfu, more preferably from about 1.10⁵ to about 1.10¹⁰ cfu andeven more preferably from about 1.10⁶ to about 1.10⁹ cfu. In anotherembodiment of the invention, the cosmetically effective amount offragments of Akkermansia muciniphila ranges from fragments derived fromabout 1.10⁶ to about 1.10¹⁰ cfu, preferably from about 1.10⁸ to about1.10¹⁰ cfu, more preferably from about 1.10⁹ to about 1.10¹⁰ cfu. Inanother embodiment of the invention, the cosmetically effective amountof fragments of Akkermansia muciniphila ranges from fragments derivedfrom about 1.10⁶ to about 1.10¹⁰ cfu, preferably from about 1.10⁶ toabout 1.10⁹ cfu, more preferably from about 1.10⁸ to about 1.10⁹ cfu.

In one embodiment of the invention, Akkermansia muciniphila or afragment thereof, or the composition or cosmetic composition isadministered at least once a week, preferably at least twice a week,more preferably at least three times a week, and even more preferablythree times a week. In another embodiment, Akkermansia muciniphila or afragment thereof, or the composition or cosmetic composition isadministered at least once a day, and preferably at least twice a day.

In one embodiment, Akkermansia muciniphila or a fragment thereof, or thecomposition or cosmetic composition of the invention is administeredduring 1 week, preferably 2, 3, 4, 5, 6, 7 or 8 weeks or more.

In one embodiment, Akkermansia muciniphila or a fragment thereof, or thecomposition or cosmetic composition of the invention is administered fora period that lasts until the desired outcome is achieved (e.g. weightloss . . . ).

In one embodiment, the administration of Akkermansia muciniphila or afragment thereof, or the composition or cosmetic composition of theinvention is permanent, i.e. is not limited in time.

In one embodiment of the invention, the daily amount of Akkermansiamuciniphila administered per day ranges from 1.10² to about 1.10¹⁵cfu/day, preferably from about 1.10⁵ to about 1.10¹² cfu/day, morepreferably from about 1.10⁸ to about 1.10¹⁰ cfu/day, and even morepreferably from about 1.10⁹ to about 1.10¹⁰ cfu/day. In anotherembodiment of the invention, the daily amount of Akkermansia muciniphilaadministered per day ranges from 1.10⁶ to about 1.10¹⁰ cfu/day,preferably from about 1.10⁸ to about 1.10¹⁰ cfu/day, more preferablyfrom about 1.10⁹ to about 1.10¹⁰ cfu/day. In another embodiment of theinvention, the daily amount of Akkermansia muciniphila administered perday ranges from 1.10⁶ to about 1.10¹⁰ cfu/day, preferably from about1.10⁶ to about 1.10⁹ cfu/day, more preferably from about 1.10⁸ to about1.10⁹ cfu/day.

In one embodiment of the invention, the daily amount of fragments ofAkkermansia muciniphila administered per day ranges from fragmentsderived from about 1.10² to about 1.10¹⁵ cfu/day, preferably from about1.10⁵ to about 1.10¹² cfu/day, more preferably from about 1.10⁸ to about1.10¹⁰ cfu/day, and even more preferably from about 1.10⁹ to about1.10¹⁰ cfu/day. In another embodiment of the invention, the daily amountof fragments of Akkermansia muciniphila administered per day ranges fromfragments derived from about 1.10⁶ to about 1.10¹⁰ cfu/day, preferablyfrom about 1.10⁸ to about 1.10¹⁰ cfu/day, more preferably from about1.10⁹ to about 1.10¹⁰ cfu/day. In another embodiment of the invention,the daily amount of fragments of Akkermansia muciniphila administeredper day ranges from fragments derived from about 1.10⁶ to about 1.10¹⁰cfu/day, preferably from about 1.10⁶ to about 1.10⁹ cfu/day, morepreferably from about 1.10⁸ to about 1.10⁹ cfu/day.

In one embodiment, said subject is not an obese subject. In anotherembodiment, said subject is overweight.

In one embodiment of the invention, the composition, the pharmaceuticalcomposition, the cosmetic composition or the medicament furthercomprises additional probiotic strains or species, such as, for example,bacterial probiotic strains or species; prokaryotes probiotics otherthan bacteria; or fungal strains or species, preferably yeast strains orspecies. In one embodiment, said additional probiotic strains or speciesare selected from those naturally present in the gut of the subject,preferably in the human gut, more preferably in the gut of healthy humansubjects.

Examples of bacterial probiotic strains or species that may be used inthe present invention include, but are not limited to Lactobacillus,Lactococcus, Bifidobacterium, Veillonella, Desemzia, Coprococcus,Collinsella, Citrobacter, Turicibacter, Sutterella, Subdoligranulum,Streptococcus, Sporobacter, Sporacetigenium, Ruminococcus, Roseburia,Proteus, Propionobacterium, Leuconostoc, Weissella, Pediococcus,Streptococcus, Prevotella, Parabacteroides, Papillibacter, Oscillospira,Melissococcus, Dorea, Dialister, Clostridium, Cedecea, Catenibacterium,Butyrivibrio, Buttiauxella, Bulleidia, Bilophila, Bacteroides,Anaerovorax, Anaerostopes, Anaerofilum, Enterobacteriaceae, Fermicutes,Atopobium, Alistipes, Acinetobacter, Slackie, Shigella, Shewanella,Serratia, Mahella, Lachnospira, Klebsiella, Idiomarina, Fusobacterium,Faecalibacterium, Eubacterium, Enterococcus, Enterobacter, Eggerthella.

Examples of prokaryote strains or species that may be used in thepresent invention include, but are not limited to Archaea, Firmicutes,Bacteroidetes (such as, for example, Allistipes, Bacteroides ovatus,Bacteroides splachnicus, Bacteroides stercoris, Parabacteroides,Prevotella ruminicola, Porphyromondaceae, and related genus),Proteobacteria, Betaproteobacteria (such as, for example, Aquabacteriumand Burkholderia), Gammaproteobacteria (such as, for example,Xanthomonadaceae), Actinobacteria (such as, for example,Actinomycetaceae and Atopobium), Fusobacteria, Methanobacteria,Spirochaetes, Fibrobacters, Deferribacteres, Deinococcus, Therms,Cyanobacteria, Methanobrevibacteria, Peptostreptococcus, Ruminococcus,Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis, Gemella,Roseburia, Dialister, Anaerotruncus, Staphylococcus, Micrococcus,Propionobacteria, Enterobacteriaceae, Faecalibacteria, Bacteroides,Parabacteroides, Prevotella, Eubacterium, Bacilli (such as, for example,Lactobacillus salivarius and related species, Aerococcus,Granulicatella, Streptococcus bovis and related genus and Streptococcusintermedius and related genus), Clostridium (such as, for example,Eubacterium hallii, Eubacterium limosum and related genus) andButyrivibrio.

Examples of fungal probiotic strains or species, preferably yeastprobiotic strains or species that may be used in the present inventioninclude, but are not limited Ascomycetes, Zygomycetes andDeuteromycetes, preferably from the groups Aspergillus, Torulopsis,Zygosaccharomyces, Hansenula, Candida, Saccharomyces, Clavispora,Bretanomyces, Pichia, Amylomyces, Zygosaccharomyces, Endomycess,Hyphopichia, Zygosaccharomyces, Kluyveromyces, Mucor, Rhizopus,Yarrowia, Endomyces, Debaryomyces, and/or Penicillium.

The Applicant herein shows that the beneficial effects observed afterAkkermansia muciniphila administration are specific of this bacterialstrain. Indeed, it is shown in the Examples that the administration ofLactobacillus plantarum WCSF-1 does not have the same beneficialeffects.

In one embodiment of the invention, the composition, the pharmaceuticalcomposition, the cosmetic composition or the medicament does notcomprise the bacterial strains Lactobacillus-Enterococcus, Bacteroidesand/or Atopobium.

In one embodiment of the invention, the only one microbial strain orspecies, preferably bacterial strain or species, comprised in thecomposition, pharmaceutical composition, cosmetic composition ormedicament is Akkermansia muciniphila.

In one embodiment of the invention, the composition, pharmaceuticalcomposition, cosmetic composition or medicament consists of Akkermansiamuciniphila.

In another embodiment of the invention, the composition, pharmaceuticalcomposition, cosmetic composition or medicament consists essentially ofAkkermansia muciniphila, wherein “consisting essentially of” hereinmeans that Akkermansia muciniphila is the only microbial strain orspecies, preferably the only bacterial strain or species comprised inthe composition, pharmaceutical composition, cosmetic composition ormedicament.

In one embodiment of the invention, Akkermansia muciniphila or afragment thereof activates or inhibits the growth and/or biologicalactivity of other bacterial strain(s) or species of the gut microbiota.

In one embodiment of the invention, the composition, the pharmaceuticalcomposition, the cosmetic composition or the medicament furthercomprises a prebiotic.

Examples of prebiotics that may be used in the present inventioninclude, but are not limited to, inulin and inulin-type fructans,oligofructose, xylose, arabinose, arabinoxylan, ribose, galactose,rhamnose, cellobiose, fructose, lactose, salicin, sucrose, glucose,esculin, tween 80, trehalose, maltose, mannose, mellibiose, mucus ormucins, raffinose, fructooligosaccharides, galacto-oligosaccharides,amino acids, alcohols, and any combinations thereof.

Other non-limiting examples of prebiotics include water-solublecellulose derivatives, water-insoluble cellulose derivatives,unprocessed oatmeal, metamucil, all-bran, and any combinations thereof.

Examples of water-soluble cellulose derivatives include, but are notlimited to, methylcellulose, methyl ethyl cellulose, hydroxyethylcellulose, ethyl hydroxyethyl cellulose, cationic hydroxyethylcellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose,hydroxypropyl methylcellulose, and carboxymethyl cellulose.

Akkermansia muciniphila or a fragment thereof or the composition,pharmaceutical composition, cosmetic composition or medicament of theinvention may be administered by several routes of administration.Examples of adapted routes of administration include, but are notlimited to, oral administration, rectal administration, administrationvia esophagogastroduodenoscopy, administration via colonoscopy,administration using a nasogastric or orogastric tube and the like.

According to an embodiment, Akkermansia muciniphila or a fragmentthereof or the composition, pharmaceutical composition, cosmeticcomposition or medicament of the invention is in a form adapted to oraladministration. According to a first embodiment, the form adapted tooral administration is a solid form selected from the group comprisingtablets, pills, capsules, soft gelatin capsules, sugarcoated pills,orodispersing/orodispersing tablets, effervescent tablets or othersolids. According to a second embodiment, the form adapted to oraladministration is a liquid form, such as, for example, a drinkablesolution, liposomal forms and the like.

In one embodiment, the composition, pharmaceutical composition, cosmeticcomposition or medicament of the invention further comprises excipients,diluent and/or carriers selected with regard to the intended route ofadministration. Examples of excipients, diluent and/or carriers include,but are not limited to, water, phosphate buffer saline, anaerobicphosphate buffer saline, sodium bicarbonate, juice, milk, yogurt, infantformula, dairy product, coloring agents, such as, for example, titanedioxide (E171), iron dioxide (E172) and brilliant black BN (E151);flavoring agents; thickeners, such as, for example, glycerolmonostearate; sweeteners; coating agents, such as, for example, refinedcolza oil, soya oil, peanut oil, soya lecithin or fish gelatin; dilutingagents, such as, for example, lactose, monohydrated lactose or starch;binding agents, such as, for example, povidone, pregelatinized starch,gums, saccharose, polyethylene glycol (PEG) 4000 or PEG 6000;disintegrating agents, such as, for example, microcrystalline celluloseor sodium carboxymethyl starch, such as, for example, sodiumcarboxymethyl starch type A; lubricant agents, such as, for example,magnesium stearate; flow agent, such as, for example, colloidalanhydrous silica, etc.

In one embodiment of the invention, the composition, pharmaceuticalcomposition, cosmetic composition or medicament is in the form of anutritional composition, i.e. comprises liquid or solid food, feed ordrinking water. In one embodiment of the invention, the composition,pharmaceutical composition, cosmetic composition or medicament is a foodproduct, such as, for example, dairy products, dairy drinks, yogurt,fruit or vegetable juice or concentrate thereof, powders, malt or soy orcereal based beverages, breakfast cereal such as muesli flakes, fruitand vegetable juice powders, cereal and/or chocolate bars,confectionary, spreads, flours, milk, smoothies, confectionary, milkproduct, milk powder, reconstituted milk, cultured milk, yoghurt,drinking yoghurt, set yoghurt, drink, dairy drink, milk drink,chocolate, gels, ice creams, cereals, reconstituted fruit products,snack bars, food bars, muesli bars, spreads, sauces, dips, dairyproducts including yoghurts and cheeses, drinks including dairy andnon-dairy based drinks, sports supplements including dairy and non-dairybased sports supplements.

In one embodiment of the invention, the composition, pharmaceuticalcomposition, cosmetic composition or medicament is in the form of a foodadditive, drink additive, dietary supplement, nutritional product,medical food or nutraceutical composition. Akkermansia muciniphila is astrictly anaerobic bacterium. Therefore, in the embodiment where viableor living strains are used, prolonged contact with oxygen should beavoided. Examples of means for avoiding prolonged contact with oxygeninclude, but are not limited to, freeze of the bacterial cells orpackaging in a sealed container and the like.

The Applicant herein showed that obesity and related disorders areassociated with an increased gut permeability and with impaired mucusproduction, epithelium barrier, immune system and/or antibacterialcompounds production by the subject; and that the administration of A.muciniphila may restore these parameters.

Therefore, the present invention also relates to Akkermansia muciniphilaor a fragment thereof for decreasing gut permeability and/or forrestoring impaired mucus production and/or for restoring epitheliumbarrier and/or for restoring immune system and/or for decreasing theproduction of antibacterial compounds. Another object of the inventionis a method for decreasing gut permeability and/or for restoringimpaired mucus production and/or for restoring epithelium barrier and/orfor restoring immune system and/or for decreasing the production ofantibacterial compounds in a subject in need thereof, comprisingadministering an effective or cosmetically effective amount ofAkkermansia muciniphila or a fragment thereof to a subject in needthereof.

The Applicant surprisingly showed that the administration of A.muciniphila controls gut barrier by regulating mucus layer thickness andthe production of colon antimicrobial peptides (such as, for example,RegIIIgamma). In addition, they also showed that A. muciniphilaregulates the production of acylglycerols that belongs to theendocannabinoids family involved in the control of inflammation, gutbarrier and gut peptides secretion (GLP-1 and GLP-2). GLP-1 and GLP-2are involved in a great variety of functions, including improvinginsulin signalling, decreasing inflammation, and promoting satiety.

Therefore, the present invention also relates to Akkermansia muciniphilaor a fragment thereof for controlling gut barrier function, and to amethod for controlling gut barrier function comprising administering aneffective or cosmetically effective amount of Akkermansia muciniphila ora fragment thereof to a subject in need thereof. In one embodiment,Akkermansia muciniphila or a fragment thereof regulates mucus layerthickness (which may be decreased in obesity or other metabolicdisorders). In another embodiment, the administration of Akkermansiamuciniphila or a fragment thereof induces the production of colonantimicrobial peptides, such as, for example, RegIIIgamma. In anotherembodiment, the administration of Akkermansia muciniphila or a fragmentthereof induces the production of compounds of the endocannabinoidsfamily, such as, for example, acylglycerols selected from the groupcomprising 2-oleoylglycerol, 2-palmitoylglycerol and2-arachidonoylglycerol. In another embodiment, the administration ofAkkermansia muciniphila or a fragment thereof regulates mucus turnover.

Another object of the invention concerns Akkermansia muciniphila or afragment thereof for use in treating metabolic dysfunction associatedwith or caused by a metabolic disorder. Still another object of theinvention is thus a method for treating metabolic dysfunction associatedwith or caused by a metabolic disorder in a subject in need thereof,comprising administering an effective amount or a cosmetically effectiveamount of Akkermansia muciniphila or a fragment thereof to a subject inneed thereof.

The Applicant also showed that the administration of Akkermansiamuciniphila controls fat storage and adipose tissue metabolism.Therefore, another object of the invention concerns Akkermansiamuciniphila or a fragment thereof for use in controlling fat storage andadipose tissue metabolism. Another object of the invention is also amethod for controlling fat storage and adipose tissue metabolismcomprising administering an effective amount or a cosmetically effectiveamount of Akkermansia muciniphila or a fragment thereof to a subject inneed thereof. In one embodiment, said control does not involve anychange in food intakes. In one embodiment of the invention,administration of Akkermansia muciniphila or a fragment thereofabolishes metabolic endotoxemia. In another embodiment, administrationof Akkermansia muciniphila or a fragment thereof lowers fat mass. Inanother embodiment, administration of Akkermansia muciniphila or afragment thereof increases mRNA expression of adipocyte differentiationand lipid oxidation, preferably without affecting lipogenesis.

The Applicant also showed that the administration of Akkermansiamuciniphila regulates adipose tissue metabolism and glucose homeostasis.The present invention thus relates to Akkermansia muciniphila or afragment thereof for use in the regulation of adipose tissue metabolismand glucose homeostasis; and to a method for regulating adipose tissuemetabolism and glucose homeostasis comprising administering an effectiveamount or a cosmetically effective amount of Akkermansia muciniphila ora fragment thereof to a subject in need thereof. In one embodiment ofthe invention, the administration of Akkermansia muciniphila or afragment thereof reverses diet-induced fasting hyperglycemia. In anotherembodiment, the administration of Akkermansia muciniphila or a fragmentthereof induces a reduction of at least 10%, preferably of at least 30%,more preferably of at least 40% of hepatic glucose-6-phosphataseexpression. In another embodiment, the administration of Akkermansiamuciniphila or a fragment thereof induces a reduction of theinsulin-resistance index. In one embodiment, said reduction of theinsulin-resistance index is of at least 5%, preferably of at least 10%,more preferably of at least 15%.

Moreover, the Applicant showed that the administration of Akkermansiamuciniphila leads to the normalization of adipose tissue CD11csubpopulation of macrophages. The amount of cells of this population ofmacrophages is increased in metabolic disorders such as, for example,obesity and Type 2 Diabetes, and is a hallmark of inflammation relatedto these metabolic disorders. Therefore, the present invention alsorelates to Akkermansia muciniphila or a fragment thereof for treatinginflammation, preferably low grade inflammation, associated with orcaused by metabolic disorders; and to a method for treating inflammationrelated to metabolic disorders comprising administering an effectiveamount or a cosmetically effective amount of Akkermansia muciniphila ora fragment thereof to a subject in need thereof. In one embodiment ofthe invention, the administration of Akkermansia muciniphila or afragment thereof decreases the amount of CD11c macrophages in theadipose tissue.

Finally, the Applicant showed that the administration of Akkermansiamuciniphila decreases plasma cholesterol in high-fat diet fed mice.Therefore, the present invention also relates to Akkermansia muciniphilaor a fragment thereof for decreasing plasma cholesterol; and to a methodfor decreasing plasma cholesterol comprising administering an effectiveamount or a cosmetically effective amount of Akkermansia muciniphila ora fragment thereof to a subject in need thereof.

In one embodiment of the invention, the administration of Akkermansiamuciniphila or a fragment thereof to a subject has no impact on foodintake of said subject.

In one embodiment of the invention, the administration of Akkermansiamuciniphila or a fragment thereof to a subject increases energyexpenditure of said subject, preferably without impacting the foodintake of said subject.

The present invention thus also relates to a method of increasing energyexpenditure of a subject, comprising administering Akkermansiamuciniphila or a fragment thereof, or a composition, pharmaceuticalcomposition, cosmetic composition or medicament of the invention to thesubject, preferably in a therapeutically or cosmetically effectiveamount. Preferably, the method of the invention does not comprise orfurther comprise modulating the food intake of said subject. In oneembodiment of the invention, the method of the invention increasesenergy expenditure, thereby inducing durable weight loss in the subject,and thereby treating metabolic disorders in said subject, such as, forexample, obesity related metabolic disorders.

In one embodiment, the administration of Akkermansia muciniphila or afragment thereof to a subject increases satiety in said subject.Consequently, according to this embodiment, the method of the inventionincreases satiety in a subject, thereby inducing durable weight loss inthe subject, and thereby treating metabolic disorders in said subject,such as, for example, obesity related metabolic disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are a combination of graphs showing that Akkermansiamuciniphila abundance is decreased in obese and diabetic mice, whereasprebiotic treatment restores it to basal levels and improves metabolicendotoxemia and related disorders. (FIG. 1A) Akkermansia muciniphilaabundance (Log₁₀ of bacteria/g of cecal content) measured in the cecalcontent of leptin-deficient (ob-ob) obese mice (n=5) and their leanlittermates (lean) (n=5). (FIG. 1B) Akkermansia muciniphila abundance(Log₁₀ of bacteria/g of cecal content) measured in the cecal content ofobese mice fed a normal chow diet (ob-CT) or treated with prebiotics(ob-Pre) for 5-weeks (n=10). (FIG. 1C) Akkermansia muciniphila abundance(Log₁₀ of bacteria per g of cecal content) measured in the cecal contentof control diet-fed mice (CT) or CT diet-fed mice treated withprebiotics (CT-Pre) added in tap water and HF diet-fed mice (HF) or HFdiet-fed mice treated with prebiotics (HF-Pre) added in tap water for8-weeks (n=10). (FIG. 1D) Portal vein serum LPS levels (n=7-9). (FIG.1E) Adipose tissue macrophages infiltration marker CD11c mRNA (n=10).(FIG. 1F) Pearson's correlation between log values of portal vein LPSlevels and Akkermansia muciniphila abundance (Log₁₀ of bacteria per g ofcecal content), inset indicates Pearson's correlation coefficient (r)and the corresponding P value. (FIG. 1G) Total fat mass gain measured bytime-domain nuclear magnetic resonance (n=10). Data in FIGS. 1A-1C areshown as boxplots. *P<0.05, by two-tailed student t-test. Data in c-ghave been obtained in the same group of mice. Data in FIGS. 1D, 1E, and1G are shown as mean±s.e.m. Data with different superscript letters aresignificantly different (P<0.05), according to the post-hoc ANOVAone-way statistical analysis.

FIGS. 2A-2F are a combination of graphs and pictures showing thatAkkermansia muciniphila changes gut microbiota composition, counteractsdiet-induced gut barrier dysfunction, changes intestinal level ofendocannabinoids and improves metabolic disorders in diet-induced obesemice. Mice were treated by daily oral gavage with Akkermansiamuciniphila (2.10⁸ bacterial cells suspended in 200 μl sterile anaerobicphosphate buffer saline (PBS)) and fed a control diet (CT-Akk) or aHF-diet (HF-Akk) compared to mice fed a control diet (CT) or a high-fatdiet (HF) and treated by daily oral gavage with an equivalent volume ofsterile anaerobic PBS for 4-weeks (n=10). (FIG. 2A) PCA analysis basedon MITChip phylogenetic fingerprints of the gut microbiota from thececal contents of control groups (CT and HF) and Akkermansia muciniphilatreated groups (CTA and HFA). (FIG. 2B) Portal vein serum LPS levels(n=6-10). (FIG. 2C) Total fat mass gain measured by time-domain nuclearmagnetic resonance (n=10). (FIG. 2D) Insulin resistance index wasdetermined by multiplying the area under the curve (from 0 min to 15min) of both blood glucose and plasma insulin obtained following an oralglucose load (2 g of glucose per kg of body weight) performed after 4weeks of treatment (n=10). (FIG. 2E) Adipose tissue macrophagesinfiltration marker CD11c mRNA (n=10). (FIG. 2F) Adipocytedifferentiation (CCAAT/enhancer—binding protein-α, encoded by Cebpa),lipogenesis (acetyl-CoA carboxylase, encoded by Acc1; fatty acidsynthase, encoded by Fasn) and lipid oxidation (carnitinepalmitoyltransferase-1, encoded by Cpt1; acyl-CoA-oxidase encoded byAcox1; peroxisome proliferator-activated receptor gamma coactivator,encoded by Pgc1a; and peroxisome proliferator-activated receptor alpha,encoded by Ppara) markers mRNA expression were measured in the visceralfat depots (mesenteric fat) n=10). (FIG. 2G) Ileum 2-palmitoylglycerol(2-PG), 2-oloeylglycerol (2-OG), 2-arachidonoylglycerol (2-AG)(expressed as % of the control) (n=10). (FIG. 2H) Mucus layer thicknessmeasured by histological analyses following alcian blue staining.Representative alcian blue images used for the mucus layer thicknessmeasurements, bars=40 μm (n=7-8). (FIG. 2I) Colon regeneratingislet-derived 3-gamma (RegIIIγ, encoded by Reg3g) mRNA expression(n=10). Data in FIG. 2A-2I have been obtained in the same group of mice.Data in FIGS. 2B-2I are shown as mean±s.e.m. Data with differentsuperscript letters are significantly different (P<0.05), according tothe post-hoc ANOVA one-way statistical analysis.

FIGS. 3A-3C are a combination of graphs and pictures showing thatprebiotic-treated mice exhibited inverse relationship betweenAkkermansia muciniphila and adipose tissue macrophage infiltration orfat mass gain. (FIG. 3A) Pearson's correlation between adipose tissueCD11c mRNA levels and Akkermansia muciniphila abundance (Log₁₀ ofbacteria per g of cecal content) measured in the cecal content ofcontrol diet-fed mice (CT) or CT diet-fed mice treated with prebiotics(CT-Pre) added in tap water and HF diet-fed mice (HF) or HF diet-fedmice treated with prebiotics (HF-Pre) added in tap water for 8-weeks,inset indicates Pearson's correlation coefficient (r) and thecorresponding P value. (FIG. 3B) Subcutaneous, mesenteric and epididymalfat depots weight (g per 100 g of body weight) (n=10). (FIG. 3C)Pearson's correlation between adipose tissue mass gain and cecal contentAkkermansia muciniphila abundance (Log₁₀ of bacteria per g of cecalcontent), inset indicates Pearson's correlation coefficient (r) and thecorresponding P value. Data in FIGS. 3A-3C have been obtained in thesame group of mice, and are shown as mean±s.e.m. Data with differentsuperscript letters are significantly different (P<0.05), according tothe post-hoc ANOVA one-way statistical analysis.

FIGS. 4A-4B are a combination of graphs showing that daily oral gavagewith Akkermansia muciniphila increases cecal abundance of thesebacteria. Akkermansia muciniphila abundance expressed as (FIG. 4A) % oftotal 16S RNA or (FIG. 4B) Log₁₀ DNA copies measured in mice treated bydaily oral gavage with Akkermansia muciniphila (2.10⁸ bacterial cellssuspended in 200 μl sterile anaerobic phosphate buffer saline (PBS)) andfed a control diet (CT-Akk) or a HF-diet (HF-Akk) compared to mice fed acontrol diet (CT) or a high-fat diet (HF) and treated by daily oralgavage with an equivalent volume of sterile anaerobic PBS for 4-weeks(n=10).

FIGS. 5A-5D are a combination of graphs and pictures showing thatAkkermansia muciniphila treatment reduces fat mass without affectingfood intake. (FIG. 5A) Subcutaneous, mesenteric and epididymal fatdepots weight (g per 100 g of body weight) of mice treated by daily oralgavage with Akkermansia muciniphila (2.10⁸ bacterial cells suspended in200 μl sterile anaerobic phosphate buffer saline (PBS)) and fed acontrol diet (CT-Akk) or a HF-diet (HF-Akk) or mice fed a control diet(CT) or a high-fat diet (HF) and treated by daily oral gavage with anequivalent volume of sterile anaerobic PBS for 4-weeks (n=10). (FIG. 5B)Cumulative food intake (g) over the 4-weeks of treatment. (FIG. 5C)Final body weight (n=10). (FIG. 5D) Final fat and lean mass expressed inpercentage of final body weight and measured using 7.5 MHz time-domainNMR (LF50 minispec; Bruker, n=10). Data are shown as mean±s.e.m. Datawith different superscript letters are significantly different (P<0.05),according to the post-hoc ANOVA one-way statistical analysis.

FIGS. 6A-6B are a combination of graphs showing that Akkermansiamuciniphila treatment normalizes fasted glycemia and reduces fastedhepatic G6pc mRNA expression. (FIG. 6A) Fasted glycemia measured in micetreated by daily oral gavage with Akkermansia muciniphila (2.10⁸bacterial cells suspended in 200 μl sterile anaerobic phosphate buffersaline (PBS)) and fed a control diet (CT-Akk) or a HF-diet (HF-Akk) ormice fed a control diet (CT) or a high-fat diet (HF) and treated bydaily oral gavage with an equivalent volume of sterile anaerobic PBS for4-weeks (n=10). (FIG. 6B) Glucose-6 phosphatase (encoded by G6pc) mRNAexpression levels measured in the liver at the end of the 4-weeks period(n=10). Data are shown as mean±s.e.m. Data with different superscriptletters are significantly different (P<0.05), according to the post-hocANOVA one-way statistical analysis.

FIGS. 7A-7E is a combination of graphs showing that Akkermansiamuciniphila treatment has minor effects on antibacterial peptidescontent in the ileum and IgA levels in the faeces. Antibacterialpeptides mRNA expression of (FIG. 7A) Regenerating islet-derived 3-gamma(RegIIIγ, encoded by Reg3g), (FIG. 7B) Phospholipase A2 group IIA(encoded by Pla2g2a), (FIG. 7C) α-defensins (encoded by Defa) and (FIG.7D) Lysozyme C (encoded by Lyz1) measured in the ileum of mice treatedby daily oral gavage with Akkermansia muciniphila (2.10⁸ bacterial cellssuspended in 200 μl sterile anaerobic phosphate buffer saline (PBS)) andfed a control diet (CT-Akk) or a HF-diet (HF-Akk) or mice fed a controldiet (CT) or a high-fat diet (HF) and treated by daily oral gavage withan equivalent volume of sterile anaerobic PBS for 4-weeks (n=10). (FIG.7E) Fecal IgA levels (μg/g of feces). Data are shown as mean s.e.m. Datawith different superscript letters are significantly different (P<0.05),according to the post-hoc ANOVA one-way statistical analysis.

FIGS. 8A-8G are a combination of histograms, graphs and images showingthat heat-inactivated A. muciniphila did not counteract metabolicendotoxemia, diet-induced obesity, oral glucose intolerance, did notimprove adipose tissue metabolism and gut barrier function indiet-induced obese mice. Control mice were fed a control (CT) or HF diet(HF) and treated with a daily oral gavage containing sterile anaerobicPBS and glycerol for 4 weeks daily. Treated mice received an oral gavageof alive A. muciniphila (HF-Akk) or metabolically inactivated A.muciniphila (HF-K-Akk) (2.10⁸ bacterial cells suspended in 200 μl ofsterile anaerobic phosphate-buffered saline (PBS)) and fed a HF diet(n=8). (FIG. 8A) Portal vein serum LPS levels (n=6-7). (FIG. 8B) Totalfat mass gain measured by time-domain nuclear magnetic resonance(n=7-8). (FIG. 8C) Plasma glucose profile following 2 g/kg glucose oralchallenge in freely moving mice, and the histogram in (FIG. 8D) showsthe mean area under the curve (AUC) measured between 0 and 120 min afterglucose load (n=7-8). (FIG. 8E) mRNA expression of markers of adipocytedifferentiation (Cebpa), lipogenesis (Acc1; Fasn) and lipid oxidation(Cpt1; Acox1; Pgc1a; and Ppara) was measured in visceral fat depots(mesenteric fat) (n=8). (FIG. 8F) Thickness of the mucus layer measuredby histological analyses following alcian blue staining (CT n=4, HF n=6,HF-Akk and HF-K-Akk n=5). (FIG. 8G) Representative alcian blue imagesthat were used for mucus layer thickness measurements, bars=40 μm.M=mucosa, IM=inner mucus layer. Data are shown as means s.e.m. Data withdifferent superscript letters are significantly different (P<0.05)according to post-hoc ANOVA one-way statistical analysis.

FIGS. 9A-9B is a combination of histograms showing that heat-inactivatedA. muciniphila did not reduce subcutaneous, mesenteric and epididymalfat mass and did not increase colon antimicrobial peptides in mice on anHF diet. (FIG. 9A) Subcutaneous, mesenteric and epididymal fat depotweights (g per 100 g body weight) measured in control mice fed a control(CT) or HF diet (HF) and treated with a daily oral gavage containingsterile anaerobic PBS and glycerol for 4 weeks daily. Treated micereceived an oral gavage of alive A. muciniphila (HF-Akk) or killed A.muciniphila (HF-K-Akk) (2.10⁸ bacterial cells suspended in 200 μl ofsterile anaerobic PBS) and fed a HF diet (n=8). (FIG. 9B) mRNAexpression of colon regenerating islet-derived 3-gamma (RegIIIγ, encodedby Reg3g) mRNA expression (n=8-18), data represents the results from thetwo A. muciniphila studies. Data are shown as means±s.e.m. Data withdifferent superscript letters are significantly different (P<0.05)according to a post-hoc ANOVA one-way statistical analysis.

FIGS. 10A-10C are a combination of histograms showing the efficiency ofa treatment with A. muciniphila 3 times a week during 8 weeks. (FIG.10A) Body weight (g) measured in control mice fed a control (CT) (n=8)or HF diet (HF) (n=10) and treated 3 times per weeks by oral gavage withsterile anaerobic PBS containing glycerol or A. muciniphila (HF-Akk)(n=10) for 8 weeks. (2.10⁸ bacterial cells suspended in 200 μl ofsterile anaerobic PBS). (FIG. 8B) Subcutaneous fat mass. (FIG. 10C)Visceral adipose tissues (mesenteric and epidydimal). Data are shown asmeans±s.e.m. Data with different superscript letters are significantlydifferent (P<0.05) according to a post-hoc ANOVA one-way statisticalanalysis.

FIG. 11 is an histogram showing that A. muciniphila reduces plasmacholesterol in mice fed a high-fat diet. (CT) Mice fed a control diet;(Akk) Mice treated by daily oral gavage with Akkermansia muciniphila(2.10⁸ bacterial cells suspended in 200 μl sterile anaerobic phosphatebuffer saline (PBS)) and fed a control diet; (HF) Mice fed a high-fatdiet; (HF-Akk) Mice treated by daily oral gavage with Akkermansiamuciniphila (10⁹ bacterial cells suspended in 200 μl sterile anaerobicphosphate buffer saline (PBS)) and fed a HF-diet.

FIGS. 12A-12E are a combination of histograms showing that L. plantarumWCFS1 did not reduce fat mass and did not improve adipose tissuemetabolism and gut barrier function in diet-induced obese mice. Controlmice were fed a control (CT) or HF diet (HF) and treated with a dailyoral gavage containing sterile anaerobic PBS and glycerol for 4 weeks.Treated mice received an oral gavage of L. plantarum WCFS1 (HF-LP)(2.10⁸ bacterial cells suspended in 200 μL of sterile anaerobic PBS) andfed a HF diet (n=7-8). (FIG. 12A) Final fat mass measured by time-domainNMR (n=7-8). s.c., mesenteric and epididymal fat depot weights (g/100 gof body weight) (n=7-8). (FIG. 12B) mRNA expression of markers ofadipocyte differentiation (Cebpa), lipogenesis (Acc1; Fasn) and lipidoxidation (Cpt1; Acox1; Pgc1a; and Ppara) was measured in visceral fatdepots (mesenteric fat) (n=7-8). (FIG. 12C) Thickness of the mucus layermeasured by histological analyses after alcian blue staining (n=4-6).(FIG. 12D) Portal vein serum LPS levels (n=6-7). (FIG. 12E) mRNAexpression of colon RegIIIγ (encoded by Reg3g) mRNA expression (n=8-18).Data are shown as means±s.e.m. Data with different superscript lettersare significantly different (P<0.05) according to post hoc ANOVA one-waystatistical analysis.

EXAMPLES

The present invention is further illustrated by the following examples.

Materials and Methods Mice

ob/ob experiment: ob/ob versus lean study: Six-week-old ob/ob(n=5/group) mice (C57BL/6 background, Jackson-Laboratory, Bar Harbor,Me., USA) were housed in a controlled environment (12-h daylight cycle,lights-off at 6-pm) in groups of two or three mice/cage, with freeaccess to food and water. The mice were fed a control diet (A04,Villemoisson-sur-Orge, France) for 16 weeks. Cecal content was harvestedimmersed in liquid nitrogen, and stored at −80° C., for furtherAkkermansia muciniphila analysis.

ob/ob prebiotic study: Six-week-old ob/ob (n=10/group) mice (C57BL/6background, Jackson-Laboratory, Bar Harbor, Me., USA) were housed in acontrolled environment (12-h daylight cycle, lights-off at 6-pm) ingroups of two mice/cage, with free access to food and water. The micewere fed a control diet (Ob-CT) (A04, Villemoisson-sur-Orge, France) ora control diet supplemented with prebiotics, such as oligofructose(Ob-Pre) (Orafti, Tienen, Belgium) for 5-weeks as previously described(Everard et al. Diabetes 60, 2775-2786 (2011)). This set of mice hasbeen previously characterized in Everard et al (Everard et al. Diabetes60, 2775-2786 (2011)).

High-Fat Prebiotic Experiment:

A set of 10-week-old C57BL/6J mice (40 mice, n=10/group) (Charles River,Brussels, Belgium) were housed in groups of five mice/cage, with freeaccess to food and water. The mice were fed a control diet (CT) (A04,Villemoisson-sur-Orge, France) or a control diet and treated withprebiotics, such as oligofructose (Orafti, Tienen, Belgium) (0.3g/mouse/day) added in tap water (CT-Pre), or fed a high-fat diet (HF)(60% fat and 20% carbohydrates (kcal/100 g), D12492, Research diet, NewBrunswick, N.J., USA) or a HF diet and treated with oligofructose (0.3g/mouse/day) added in tap water (HF-Pre). Treatment continued for 8weeks.

HFD Akkermansia muciniphila Treatment:

A set of 10-week-old C57BL/6J mice (40 mice, n=10/group) (Charles River,Brussels, Belgium) were housed in groups of 2 mice/cage, with freeaccess to food and water. The mice were fed a control diet (CT)(AIN93Mi; Research diet, New Brunswick, N.J., USA) or a high-fat diet(HF) (60% fat and 20% carbohydrates (kcal/100 g), D12492, Research diet,New Brunswick, N.J., USA). Mice were treated with an oral administrationof Akkermansia muciniphila by oral gavage at the dose 2.10⁸ cfu/0.2 mlsuspended in sterile anaerobic phosphate buffer saline (CT-Akk andHF-Akk) and control groups were treated with an oral gavage of anequivalent volume of sterile anaerobic phosphate buffer saline (CT andHF). Treatment continued for 4 weeks.

A. muciniphila Muc^(T) (ATTC BAA-835) was grown anaerobically in amucin-based basal medium as previously described (Derrien et al Int JSyst Evol Microbiol 54, 1469-1476 (2004)) and then washed and suspendedin anaerobic phosphate buffer saline, including 25% (v/v) glycerol, toan end concentration of 1.10¹⁰ cfu/ml.

HFD Akkermansia muciniphila alive treatment vs heat-killed andLactobacillus platarum WCFS1: A set of 10-week-old C57BL/6J mice (40mice, n=8/group) (Charles River, Brussels, Belgium) were housed ingroups of 2 mice/cage, with free access to food and water. The mice werefed a control diet (CT) (AIN93Mi; Research diet, New Brunswick, N.J.,USA) or a high-fat diet (HF) (60% fat and 20% carbohydrates (kcal/100g), D12492, Research diet, New Brunswick, N.J., USA). Mice were treateddaily with an oral administration of Akkermansia muciniphila by oralgavage at the dose 2.10⁸ cfu/0.2 ml suspended in sterile anaerobicphosphate buffer saline A. muciniphila was heat-killed/inactivated byautoclaving (15 min, 121° C., 225 kPa). A viability check by culturingon mucin-containing medium confirmed the absence of any viable cells.Lactobacillus plantarum WCFS1 was grown anaerobically in MRS medium(Difco Lactobacilli MRS broth; BD), washed, concentrated, andmanipulated an identical ways as the A. muciniphila preparation. The twocontrols group (CT and HF) were treated daily with an oral gavage of anequivalent volume of sterile anaerobic PBS containing a similar endconcentration of glycerol (2.5%) (reduced with one drop of 100 mMtitanium citrate) as the treatment groups for 4 weeks.

Food and water intake were recorded once a week. Body composition wasassessed by using 7.5 MHz time domain-nuclear magnetic resonance(TD-NMR) (LF50 minispec, Bruker, Rheinstetten, Germany).

All mouse experiments were approved by and performed in accordance withthe guidelines of the local ethics committee. Housing conditions werespecified by the Belgian Law of Apr. 6, 2010, regarding the protectionof laboratory animals (agreement number LA1230314).

Tissue Sampling

The animals have been anesthetized with isoflurane (Forene®, Abbott,Queenborough, Kent, England) before exsanguination and tissue sampling,then mice were killed by cervical dislocation. Adipose depots(epididymal, subcutaneous and mesenteric), liver were preciselydissected and weighed; the addition of the three adipose tissuescorresponds to the adiposity index. The intestinal segments (ileum,cecum and colon), the cecal content and the adipose tissues wereimmersed in liquid nitrogen, and stored at −80° C., for furtheranalysis.

Mucus Layer Thickness

Proximal colon segments were immediately removed and fixed in Carnoy'ssolution (ethanol-acetic acid-chloroform, 6/3/1 v/v/v) for two hours at4° C. Then the samples were immersed in ethanol 100% for 24 hours priorto processing for paraffin embedding. Paraffin sections of 5 μm werestained with alcian blue. A minimum of 20 different measurements weremade perpendicular to the inner mucus layer per field by an investigatorblinded to the experimental groups. 5 to 19 randomly selected fieldswere analyzed for each colon for a total of 2146 measurements by usingan image analyzer (Motic-image Plus 2.0 ML, Motic, China).

RNA Preparation and Real-Time qPCR Analysis

Total RNA was prepared from tissues using TriPure reagent (Roche).Quantification and integrity analysis of total RNA was performed byrunning 1 μl of each sample on an Agilent 2100 Bioanalyzer (Agilent RNA6000 Nano Kit, Agilent).

cDNA was prepared by reverse transcription of 1 μg total RNA using aReverse Transcription System kit (Promega, Leiden, The Netherlands).Real-time PCRs were performed with the StepOnePlus™ real-time PCR systemand software (Applied Biosystems, Den Ijssel, The Netherlands) usingMesa Fast gPCR™ (Eurogentec, Seraing, Belgium) for detection accordingto the manufacturer's instructions. RPL19 was chosen as the housekeepinggene. All samples were run in duplicate in a single 96-well reactionplate, and data were analyzed according to the 2^(ΔCT) method. Theidentity and purity of the amplified product was checked throughanalysis of the melting curve carried out at the end of amplification.Primer sequences for the targeted mouse genes are presented in the Table1 below.

Primers Sequence RPL-19 Forward GAAGGTCAAAGGGAATGTGTTCA (SEQ ID NO: 1)Reverse CCTGTTGCTCACTTGT (SEQ ID NO: 2) Reg3g ForwardTTCCTGTCCTCCATGATCAAA (SEQ ID NO: 3) Reverse CATCCACCTCTGTTGGGTTC(SEQ ID NO: 4) Lyz1 Forward GCCAAGGTCTACAATCGTTGTGAGTTG (SEQ ID NO: 5)Reverse CAGTCAGCCAGCTTGACACCACG (SEQ ID NO: 6) Pla2g2a ForwardAGGATTCCCCCAAGGATGCCAC (SEQ ID NO: 7) Reverse CAGCCGTTTCTGACAGGAGTTCTGG(SEQ ID NO: 8) CD11cc Forward ACGTCAGTACAAGGAGATGTTGGA (SEQ ID NO: 9)Reverse ATCCTATTGCAGAATGCTTCTTTACC (SEQ ID NO: 10) Defa ForwardGGTGATCATCAGACCCCAGCATCAGT (SEQ ID NO: 11) ReverseAAGAGACTAAAACTGAGGAGCAGC (SEQ ID NO: 12) Fasn ForwardTTCCAAGACGAAAATGATGC (SEQ ID NO: 13) Reverse AATTGTGGGATCAGGAGAGC(SEQ ID NO: 14) Cpt1a Forward AGACCGTGAGGAACTCAAACCTAT (SEQ ID NO: 15)Reverse TGAAGAGTCGCTCCCACT (SEQ ID NO: 16) Pgc1a ForwardAGCCGTGACCACTGACAACGAG (SEQ ID NO: 17) Reverse GCTGCATGGTTCTGAGTGCTAAG(SEQ ID NO: 18) Ppara Forward CAACGGCGTCGAAGACAAA (SEQ ID NO: 19)Reverse TGACGGTCTCCACGGACAT (SEQ ID NO: 20) Acox1 ForwardCTATGGGATCAGCCAGAAAGG (SEQ ID NO: 21) Reverse AGTCAAAGGCATCCACCAAAG(SEQ ID NO: 22) Acc1 Forward TGTTGAGACGCTGGTTTGTAGAA (SEQ ID NO: 23)Reverse GGTCCTTATTATTGTCCCAGACGTA (SEQ ID NO: 24) Cebpa ForwardGAGCCGAGATAAAGCCAAACA (SEQ ID NO: 25) Reverse GCGCAGGCGGTCATTG(SEQ ID NO: 26) G6pc Forward AGGAAGGATGGAGGAAGGAA (SEQ ID NO: 27)Reverse TGGAACCAGATGGGAAAGAG (SEQ ID NO: 28)

Insulin Resistance Index

Insulin resistance index was determined by multiplying the area underthe curve (0 min and 15 min) of both blood glucose and plasma insulinobtained following an oral glucose load (2 g of glucose per kg of bodyweight) performed after 4 weeks (A. muciniphila study) of treatment.Food was removed two-hours after the onset of the daylight cycle andmice were treated after 6-h-fasting period as previously described(Everard et al. Diabetes 60, 2775-2786 (2011)).

Biochemical Analyses

Portal vein blood LPS concentration was measured by using Endosafe-MCS(Charles River Laboratories, Lyon, France) based on the Limulusamaebocyte Lysate (LAL) kinetic chromogenic methodology that measurescolor intensity directly related to the endotoxin concentration in asample. Serum were diluted 1/10 with endotoxin free buffer to minimizeinterferences in the reaction (inhibition or enhancement) and heated 15min at 70° C. Each sample was diluted 1/70 or 1/100 with endotoxin-freeLAL reagent water (Charles River Laboratories) and treated in duplicateand two spikes for each sample were included in the determination. Allsamples have been validated for the recovery and the coefficientvariation. The lower limit of detection was 0.005 EU/ml. Plasma insulinconcentration was determined in 25 μl of plasma using an ELISA kit(Mercodia, Upssala, Sweden) according to the manufacturer instructions.

Fecal IgA levels were determined using an ELISA kit (E99-103, BethylLaboratories, Montgomery, Tex.). Freshly collected feces were frozen at−80° C. then diluted in 50 mM Tris, pH 7.4, 0.14M NaCl, 1% bovine serumalbumin, 0.05% Tween 20. A 1/250 dilution was used to measure IgA byELISA following the manufacturer instructions.

DNA Isolation from Mouse Cecal Samples

The cecal content of mice collected post mortem was stored at −80° C.Metagenomic DNA was extracted from the cecal content using a QIAamp-DNAstool mini-kit (Qiagen, Hilden, Germany) according to manufacturer'sinstructions.

Measurement of Endocannabinoids Intestinal Levels

Ileum tissues were homogenised in CHCl₃ (10 ml), and deuteratedstandards were added. The extraction and the calibration curves weregenerated as previously described (Muccioli et al, Mol Syst Biol, 2010,6, 392), and the data were normalised by tissue sample weight.

qPCR: Primers and Conditions

The primers and probes used to detect Akkermansia muciniphila were basedon 16S rRNA gene sequences: F-Akkermansia muciniphilaCCTTGCGGTTGGCTTCAGAT (SEQ ID NO: 29), R-Akkermansia muciniphilaCAGCACGTGAAGGTGGGGAC (SEQ ID NO: 30). Detection was achieved withStepOnePlus™ real-time PCR system and software (Applied Biosystems, DenIjssel, The Netherlands) using Mesa Fast gPCR™ (Eurogentec, Seraing,Belgium) according to the manufacturer's instructions Each assay wasperformed in duplicate in the same run. The cycle threshold of eachsample was then compared to a standard curve (performed in triplicate)made by diluting genomic DNA (five-fold serial dilution) (DSMZ,Braunshweig, Germany). The data are expressed as Log of bacteria/g ofcecal content.

MITChip: PCR Primers and Conditions

DNA extracted from cecal contents was analyzed using the MouseIntestinal Tract Chip (MITChip), a phylogenetic microarray consisting of3,580 different oligonucleotides probes targeting two hypervariableregions of the 16S rRNA gene (V1 and V6 regions). Analysis of theMITChip were performed as previously described (Everard et al. Diabetes60, 2775-2786 (2011); Geurts et al. Front Microbiol. 2, 149 (2011)).Briefly, Microbiota analysis was carried out on level 2 corresponding togenus-like level. Multivariate analysis was performed byrepresentational difference analysis (RDA) as implemented in the CANOCO4.5 software package (Biometris, Wageningen, The Netherlands) on averagesignal intensities for 99 bacterial groups (levels 2). All environmentalvariables were transformed as log (1+X). A Monte-Carlo permutation testbased on 999 random permutations was used to test the significance. Pvalues<0.05 were considered significant.

Measurement of Plasma Cholesterol

Plasma samples were assayed for cholesterol by measuring cholesterolpresent after enzymatic hydrolysis of ester cholesterol, using acommercial kit (DiaSys, Condom, France).

Statistical Analysis

Data are expressed as means±s.e.m. Differences between two groups wereassessed using the unpaired two-tailed Student's t-test. Data setsinvolving more than two groups were assessed by ANOVA followed byNewman-Keuls post hoc tests. Correlations were analyzed using Pearson'scorrelation. Data with different superscript letters are significantlydifferent P<0.05, according to the post-hoc ANOVA statistical analysis.Data were analyzed using GraphPad Prism version 5.00 for windows(GraphPad Software, San Diego, Calif., USA). Results were consideredstatistically significant when P<0.05.

Results

We found that abundance of A. muciniphila was 3300-fold lower inleptin-deficient obese mice versus their lean littermates (FIG. 1a ).Consistently, we found a 100-fold decrease in high-fat (HF)-fed mice(FIG. 1c ). In both models, prebiotics completely restore A. muciniphilacount (FIG. 1b,c ). In HF-fed mice, prebiotics abolished metabolicendotoxemia (FIG. 1d ), normalized adipose tissue CD11c subpopulation ofmacrophages (FIG. 1e ) and lowered fat mass (FIG. 1g and FIG. 3b ).These results were significantly and inversely correlated with A.muciniphila (FIG. 1f and FIG. 3a,c ). Nevertheless, it remained todemonstrate whether molecular mechanisms underlying the onset of thesedisorders rely on the lack of A. muciniphila and in contrast if theimprovement after prebiotic treatment results from its higher abundance.

To address this question, A. muciniphila was orally administered tocontrol or HF-fed mice during four weeks, thereby increasing A.muciniphila (FIG. 4a,b ). By using a phylogenetic-microarray (MITChip)(Everard et al. Diabetes 60, 2775-2786 (2011); Geurts et al. FrontMicrobiol. 2, 149 (2011)), we found that both HF-diet and A. muciniphilacolonization significantly affected the complex relationship amongbacteria within the gut microbiota composition, as shown by principalcomponent analyses (PCA) (FIG. 2a ) and supported by relative changes ofdifferent taxa.

We found that A. muciniphila treatment normalized diet-induced metabolicendotoxemia, adiposity and adipose tissue CD11c marker (FIG. 2 b,c,e andFIG. 5a ), without any changes in food intake (FIG. 5b ). Moreover, A.muciniphila treatment reduced body weight and improved body composition(i.e. fat mass/lean mass ratio) (FIGS. 5c and 5d ). Accordingly, wehypothesized that A. muciniphila would impact on adipose tissuemetabolism, and found that under HF-diet, A. muciniphila increased mRNAexpression of markers of adipocyte differentiation and lipid oxidationwithout affecting lipogenesis (FIG. 2f ). Together, these data furthersuggest that A. muciniphila controls fat storage and adipose tissuemetabolism.

We next discovered that colonization with A. muciniphila completelyreversed diet-induced fasting hyperglycemia, by a mechanism associatedwith a 40% reduction of hepatic glucose-6-phosphatase expression (FIG.6a,b ), thereby suggesting reduced gluconeogenesis. Notably, theinsulin-resistance index was similarly reduced after treatment (FIG. 2d). Collectively, these results suggest that A. muciniphila contributesto regulation of adipose tissue metabolism and glucose homeostasis. Oneexplanation would be that A. muciniphila plays key roles at differentlevels of the regulation of gut barrier function. Recent data suggestthat intestinal cells also contribute to the maintenance of gut barrierby secreting antimicrobial peptides thereby shaping microbialcommunities (Pott et al, EMBO Rep (2012)).

To further elucidate how A. muciniphila colonization affects gut barrierfunction; we measured the expression of Paneth and epithelial cellsantibacterial markers in the ileum. We found that A. muciniphilaincreased Reg3g (regenerating islet-derived 3-gamma, RegIIIγ) expressionunder control diet, whereas this effect was not observed in HF-fed mice(FIG. 7a ). Pla2g2a, Defa expression were similar between groups,whereas Lyz1 expression tends to be lower after bacteria administration(FIG. 7 b,c,d). IgA are secreted in intestinal lumen and are known torestrict mucosal bacterial penetration (Vaishnava, S., et al. Science334, 255-258 (2011)), here we found that fecal IgA levels were notaffected by the treatments (FIG. 7e ). Thereby, suggesting that A.muciniphila controls gut barrier function by other mechanisms involvingits epithelial signalling (Derrien et al. Front Microbiol 2, 166(2011)). We may not rule out that the endocannabinoid system plays acrucial role in this context; since we found that A. muciniphilatreatment increased 2-oleoylglycerol, 2-palmitoylglycerol and2-arachidonoylglycerol intestinal levels (FIG. 2g ). Importantly,2-oleoylglycerol has been shown to stimulate glucagon-like peptide-1(GLP-1) release from intestinal L-cells suggesting that both GLP-1 andGLP-2 might improve gut barrier and glucose homeostasis in this context(Hansen, et al. J Clin Endocrinol Metab 96, E1409-1417 (2011)).Moreover, 2-arachidonoylglycerol reduces gut permeability and2-palmitoylglycerol (Alhouayek et al FASEB J 25, 2711-2721 (2011);Ben-Shabat, et al. Eur. J. Pharmacol. 353, 23-31 (1998)) potentiates2-arachidonoylglycerol anti-inflammatory effects. Therefore, it islikely that the increased levels of these three endocannabinoidsobserved after A. muciniphila colonization constitutes a molecular eventlinking these metabolic features.

Recent evidences support that interactions between gut microbiota andmucus layer are dynamic systems affecting the biology of mucus barrier(Belzer et al, ISME J 6, 1449-1458 (2012); Johansson et al, Proc NatlAcad Sci USA 108 Suppl 1, 4659-4665 (2011)). Thus we investigated theimpact of A. muciniphila colonization on the inner mucus layerthickness. Remarkably, we found a 46% thinner mucus layer in HF-fed mice(FIG. 2h ), whereas A. muciniphila colonization counteracts thisdecrease (FIG. 2h ). Together these novel findings support the idea thatthe presence of A. muciniphila within mucus layer is a crucial mechanismto control mucus turnover (Belzer et al, ISME J 6, 1449-1458 (2012)). Wenext examined whether A. muciniphila also affects Reg3g expression inthe colon epithelial cells. Strikingly, Reg3g expression was reduced byabout 50% under HF-diet. A. muciniphila completely blunted this effectand even more increased Reg3g expression by 400% as compared to HF-fedmice (FIG. 2i ). Thus this links the colonization of the colon, but notileum, by A. muciniphila with the fundamental immune mechanism by whichRegIIIγ promotes host-bacterial mutualism and regulates the spatialrelationships between microbiota and host (Vaishnava, S., et al. Science334, 255-258 (2011)). It is important to note that in germ-free mice A.muciniphila induces gene expression in the colon rather than the ileum(Derrien et al. Front Microbiol 2, 166 (2011)).

To further demonstrate whether A. muciniphila has to be alive to exertits metabolic effects, we have compared the impact of viable A.muciniphila administration (2.10⁸ bacterial cells suspended in 200 μl ofsterile anaerobic phosphate-buffered saline (PBS)) toheat-killed/inactivated A. muciniphila (autoclaving, 15 minutes, 121°C., 225 kPa). We found that viable and metabolically active A.muciniphila counteracted diet-induced metabolic endotoxemia, fat massdevelopment and altered adipose tissue metabolism (FIG. 8A, B, D andFIG. 9A) to a similar extent as observed in the first set ofexperiments. Importantly, these effects were not observed following theadministration of heat-inactivated A. muciniphila (FIG. 8A, B, D andFIG. 9A). In addition, we found that metabolically active A. muciniphilasignificantly reduced plasma glucose levels following an oral glucosetolerance test (FIG. 8C), whereas heat-inactivated A. muciniphilaexhibited similar glucose intolerance than HF-fed mice (FIG. 8C).Finally, we confirmed that metabolically active A. muciniphila restoredmucus layer thickness upon HF-diet whereas we found thatheat-inactivated A. muciniphila did not improve mucus layer thickness ascompared to HF (FIGS. 8E and F). It is worth noting that we found100-fold more metabolically active A. muciniphila recovered from thececal and colonic content of A. muciniphila treated mice as compared toHF and heat-inactivated bacteria group (HF-Akk: 9.5+/−1.02 Log₁₀cells/mg of content, HF and HF-K-Akk: 6.8+/−0.51 Log₁₀ cells/mg ofcontent, P=0.0059), thereby evidencing the viability of A. muciniphilaafter oral administration.

These results thus confirm that that HF diet-induced obesity isassociated with changes in gut microbiota composition, however,antimicrobial peptides in the ileum were not affected by the treatments.In contrast, Reg3g expression in colon epithelial cells wassignificantly reduced by approximately 50% in HF and heat-inactivated A.muciniphila treated mice, whereas metabolically active A. muciniphilatreatment completely blunted this effect and increased Reg3g expressionupon HF diet (FIG. 9B).

We then wanted to test if A. muciniphila administration was stillefficient during a prolonged high-fat diet treatment (8 weeks) and ifthe administration of A. muciniphila 3 times a week (instead of daily)was sufficient to protect against diet-induced obesity. The preparationsas well as the doses of A. muciniphila were similar to those presentedin the protocol using daily oral gavage or metabolically inactivated A.muciniphila.

We found that A. muciniphila treatment reduces body weight gain (FIG.10A) by about 30% although mice were ingesting high-fat diet without anyfat lost in their feces and changes in food intake behavior. This wasalso associated with a reduction of about 45% of the adipose tissueweight (subcutaneous adipose tissue) (FIG. 10B) and 35% decrease invisceral fat depots (mesenteric and epididymal) (FIG. 10C). Thus, thisset of data support the fact that A. muciniphila administration remainsefficient during a prolonged treatment and the treatment is stillefficient if A. muciniphila is administered 3 times per week instead ofdaily.

In order to confirm that these results were specific of A. muciniphila,we then treated HF-fed mice with a probiotic (i.e. Lactobacillusplantarum WCFS1). We found that L. plantarum administration did notchange fat mass development, adipose tissue metabolism, mucus layerthickness, colon Reg3g mRNA, and metabolic endotoxemia (FIG. 12A-E).

Hypercholesterolemia is known as a key factor involved in cardiovasculardiseases. We therefore tested the effect of A. muciniphilaadministration on plasma cholesterol of mice fed a high-fat diet. Asshown in FIG. 11, A. muciniphila treatment significantly decreases(about 15%) plasma cholesterol, thereby contributing with LPS and theother metabolic parameters to an improved cardio-metabolic risk profile.

In summary, our findings not only provide substantial insights into theintricate mechanisms by which A. muciniphila regulates the crosstalkbetween the host and the gut microbiota, but also provide a rationalefor considering the development of a treatment using this humanmucus-colonizer for the prevention or the treatment of obesity andassociated metabolic disorders such as, for example,hypercholesterolemia.

1-17. (canceled)
 18. A pharmaceutical composition for alteringmicrobiota comprising: a therapeutically effective amount of asubstantially purified Akkermansia, wherein the substantially purifiedAkkermansia comprises at least 50% of a strain of Akkermansia, aprebiotic, and a pharmaceutically acceptable carrier, wherein thepharmaceutical composition is formulated for oral delivery andencapsulated by a coating, wherein the coating does not fully degradeuntil after it exits the stomach of a subject.
 19. The pharmaceuticalcomposition of claim 18, further comprising at least one of asubstantially purified Bacteroidetes, a substantially purifiedFirmicutes and a substantially purified Proteobacteria.
 20. Thepharmaceutical composition of claim 19, wherein the substantiallypurified Bacteroidetes is Bacteroidales, the substantially purifiedFirmicutes is Clostridiales and the substantially purifiedProteobacteria is Enterobacteriales.
 21. The pharmaceutical compositionof claim 19, wherein the substantially purified Bacteroidetes isAlistipes, the substantially purified Firmicutes is Clostridium and thesubstantially purified Proteobacteria is Escherichia.
 22. Thepharmaceutical composition of claim 18, wherein the pharmaceuticalcomposition alters the relative abundance of at least one ofBacteroidetes, Verrucomicrobia, Firmicutes, Tenericutes, andProteobacteria in a gastrointestinal tract of a subject.
 23. Thepharmaceutical composition of claim 18, wherein the prebiotic is anon-digestible oligosaccharide.
 24. The pharmaceutical composition ofclaim 23, wherein the non-digestible oligosaccharide is afructooligosaccharide, a glucooligosaccharide, a xylooligosaccharide, agalactooligosaccharide, an arabinoxylan, an arabinogalactan, agalactomannan, a polydextrose, an oligofructose, inulin, and/or aderivative thereof.
 25. The pharmaceutical composition of claim 23,wherein the non-digestible oligosaccharide is a fructooligosaccharide.26. The pharmaceutical composition of claim 18, wherein the prebiotic isinulin.
 27. The pharmaceutical composition of claim 18, wherein thepharmaceutical composition is formulated for delivery to a smallintestine, a large intestine, an ileum, a cecum, or a colon region of asubject.
 28. The pharmaceutical composition of claim 27, wherein thepharmaceutical composition is formulated for delivery to an ileum or acolon region of a subject.
 29. The pharmaceutical composition of claim18, further comprising substantially purified Firmicutes.
 30. Thepharmaceutical composition of claim 29, wherein the substantiallypurified Firmicutes is substantially purified Clostridiales.
 31. Thepharmaceutical composition of claim 30, wherein the pharmaceuticalcomposition comprises between about 30% and about 60% substantiallypurified Clostridiales.
 32. The pharmaceutical composition of claim 29,wherein the substantially purified Firmicutes is substantially purifiedClostridium.
 33. The pharmaceutical composition of claim 32, wherein thepharmaceutical composition comprises between about 30% and about 50%substantially purified Clostridium.
 34. The pharmaceutical compositionof claim 18, wherein the pharmaceutical composition increases a relativeabundance of Clostridium in a subject.
 35. The pharmaceuticalcomposition of claim 18, wherein the pharmaceutical compositionincreases glucose metabolism in a subject.
 36. The pharmaceuticalcomposition of claim 18, wherein the pharmaceutical compositionincreases energy expenditure in a subject.
 37. The pharmaceuticalcomposition of claim 18, wherein the Akkermansia is lyophilized.
 38. Thepharmaceutical composition of claim 18, wherein if the compositioncomprises a mixture of bacterial strains, then at least 50% of thebacterial strains in the composition are Verrucomicrobia, Bacteroidetes,Firmicutes, or Proteobacteria.
 39. The pharmaceutical composition ofclaim 18, wherein the Akkermansia is viable.
 40. The pharmaceuticalcomposition of claim 18, wherein the pharmaceutical compositioncomprises two or more bacterial strains, wherein the two or morebacterial strains exhibit a synergistic effect in the pharmaceuticalcomposition.
 41. A pharmaceutical composition for altering microbiotacomprising: a therapeutically effective amount of a substantiallypurified Akkermansia, wherein the substantially purified Akkermansiacomprises at least 50% of a strain of Akkermansia, a prebiotic, and apharmaceutically acceptable carrier, wherein the pharmaceuticalcomposition is formulated for oral delivery and encapsulated by acoating.
 42. The pharmaceutical composition of claim 41, furthercomprising at least one of a substantially purified Bacteroidetes, asubstantially purified Firmicutes and a substantially purifiedProteobacteria.
 43. The pharmaceutical composition of claim 41, whereinthe pharmaceutical composition alters the relative abundance of at leastone of Bacteroidetes, Verrucomicrobia, Firmicutes, Tenericutes, andProteobacteria in a gastrointestinal tract of a subject.
 44. Thepharmaceutical composition of claim 41, wherein the prebiotic is anon-digestible oligosaccharide selected from the group consisting afructooligosaccharide, a glucooligosaccharide, a xylooligosaccharide, agalactooligosaccharide, an arabinoxylan, an arabinogalactan, agalactomannan, a polydextrose, an oligofructose, inulin, and/or aderivative thereof.
 45. The pharmaceutical composition of claim 41,wherein the pharmaceutical composition is formulated for delivery to asmall intestine, a large intestine, an ileum, a cecum, or a colon regionof a subject.
 46. The pharmaceutical composition of claim 41, whereinthe pharmaceutical composition increases glucose metabolism or energyexpenditure in a subject.
 47. The pharmaceutical composition of claim41, wherein the Akkermansia is viable or non-viable.